Planar lipid bilayer membranes can provide an environment that allows single-molecule electrophysiological observations of membrane channel and pore proteins (Miller, Ion Channel Reconstitution. ed. Plenum Press: New York, 1986, p. 577). Such measurements are essential to understanding the proteins' biological function, as well as the basis of highly specific sensors capable of chemical detection (Bayley et al., Nature 2001, 413(6852):226-230) or potentially sequencing DNA at the single-molecule level (Kasianowicz et al., Proc Natl Acad Sci USA 1996, 93:13770-13773; Nakane et al., J Phys Condensed Matter 2003, 15(32):R1365-R1393). Unfortunately, the physical properties of current technology planar lipid bilayer membranes limit their scientific and technological application: they are difficult to form, physically weak, subject to mechanical and acoustical perturbation, and short lived.
A recent approach to address the fragility and short lifetime of lipid bilayers has been to tether them to solid surfaces (see Knoll, et al., Rev Mol Biotech 2000, 74(3): 137-158). Such systems combine the lipid bilayer membranes' fluidity and capacity for protein incorporation with the mechanical stability of a solid support. While electrical transport through ensembles of membrane protein channels have been measured in such systems (Favero et al., Analyt Chim 2002, 460(1):23-34; Naumowicz et al., Bioelectrochem 2003, 61:21-27) no single channels have been detected to date. This is because tethered bilayer membranes have thus far proved incapable of producing the highly insulating seals necessary for single-molecule measurements (Rehak et al., The Analyst 2004, 129:1014-1025), although this is improving (Terrattaz et al., Langmuir 2003, 19:5567-5569). Studies with tethered bilayers have also shown an inability to quantitatively measure the magnitude of incorporated channel conductances, as a result of the high in-plane resistance of the electrolyte reservoir near the substrate (Krishna et al., Langmuir 2003, 19:2294-2305). Furthermore, the presence of the solid surface (typically a gold electrode) makes long-term DC measurements and analyte transport across the channel problematic.
There have been attempts to use gels to support membranes. In these cases, lipid solutions were deposited on top of pre-cast gels. The resultant membranes were, however, too leaky for single channel measurement (Kuhner et al., Biophys J 1994, 67(1):217-226; Lu et al., Bioelectrochem Bioenergetics 1996, 39:285-289; Anrather et al., J Nanosci Nanotech 2004, 4(1/2):1-22). Ide and Yanagida formed high resistance free-standing membranes self-assembled in aqueous solution and brought them into contact with a pre-cast gel on one side (Ide et al., Biochem Biophys Res Commun 1999, 265:595-599; Ide et al., Japanese J Physiol 2002, 52:429-434). Although single channels were measured in that work, the membranes still suffered from short lifetimes. Peterson and coworkers physically sandwiched a lipid membrane between two pre-formed slabs of gel (Costello et al., Biosensors Bioelectronics 1999, 14(3):265-271; Beddow et al., Anal Chem 2004, 76(8):2261-2265). Their technique, however, has not been shown to achieve sufficiently high membrane resistances for single-molecule measurements. As noted, these techniques all rely on pre-cast gels, and they have met with limited success.
Most microfluidic approaches to ion channel analysis have adapted patch clamp technologies, which require expensive and time-consuming cell culture conditions (Ionescu-Zanefti et al., Proc Natl Acad Sci USA 2005, 102: 9112-9117; Matthews and Judy, J MEMS 2006, 15: 214-222). Furthermore, cell-based systems cannot isolate and control the environment of an ion channel to the extent possible in a cell-free system and cannot be used in ion channel-based molecular sensor applications. In this context, free-standing lipid bilayers are more appealing. In a technique commonly used to fabricate free-standing lipid bilayers in the laboratory, lipids dissolved in an organic solvent are manually “painted” over an orifice in a hydrophobic sheet submerged in an aqueous solution. This lipid solution spontaneously thins to form a bilayer membrane (Mueller et al., Nature 1962, 194: 979-980; White, S. H. In Ion Channel Reconstitution; Miller, C., Ed.; Plenum Press: New York, 1986; pp 115-139). Other approaches to microfluidic lipid membrane formation have adapted this technique to microfabricated orifices in microfluidic channels made from poly(methyl methacrylate) (Suzuki et al., Langmuir 2006, 22: 1937-1942; Sandison and Morgan, J Micromech Microeng 2005, 15: S139-S144) but suffer from a number of disadvantages. Attention from an operator is necessary to manipulate the device during membrane formation. These systems rely on a three-phase interface of lipid solution, aqueous buffer, and air; air can be problematic in microfluidic systems. Finally, the solvent in the membrane precursor solution forms an annulus around the membrane that limits the degree to which the membrane can be miniaturized (White, S. H. In Ion Channel Reconstitution; Miller, C., Ed.; Plenum Press: New York, 1986; pp 115-139).
In light of the important role lipid bilayers play in a vast variety of research applications, as well as the current problems associated with producing, stabilizing, and using such bilayers, what is needed in the art are methods and devices for the production, stabilization, and use of bilayers and membranes. Disclosed herein are compositions, methods, and devices that meet these and other needs.